Phosphorothioate antisense heparanase oligonucleotides

ABSTRACT

The present invention provides antisense phosphorothioate oligonucleotides that specifically inhibit the translation of heparanase. The invention also provides various methods of reducing angiogenesis and metastasis of tumors in a subject using said antisense phosphorothioate oligonucleotides. Finally the invention provides pharmaceutical compositions comprising the said antisense phosphorothioate oligonucleotides as active ingredients.

This invention is a continuation-in-part and claims priority of U.S.Ser. No. 09/899,440, filed Jul. 5, 2001, the contents of which arehereby incorporated by reference into this application.

BACKGROUND OF THE INVENTION

Heparanase

Cancer is the second leading cause of death in the United States. Whencancer has metastasized, it can only be cured by systemic therapy,usually cytotoxic chemotherapy. Alternative methods to prevent tumorspread that would avoid cytotoxic chemotherapy are very desirable.

One area of promise in alternative methods of therapy involves the studyof heparanase. Heparanase breaks down heparan—a component of the cellsurface and extracellular matrix. It has recently been shown thatinhibition of heparanase reduces tumor spread (Kussie, et al. 1999 andVlodasky, 1999), reducing both tumor neogenesis and angiogenesis.

Antisense Phosphorothioate Oligonucleotides

One way to achieve therapeutically useful targeted inhibition of proteinexpression is likely going to be through the use of antisenseoligonucleotides. Antisense oligonucleotides are small fragments of DNAcomplementary to a defined sequence on a specified mRNA. The antisenseoligonucleotide specifically binds to targets on the mRNA molecule andin doing so inhibits the translation of a specific mRNA into protein.

Antisense oligonucleotide molecules synthesized with a phosphorothioatebackbone have proven particularly resistant to exonuclease damagecompared to standard deoxyribonucleic acids, and so they are used inpreference.

The present study discloses that instead of inhibiting heparanaseitself, another method to reduce tumor spread may be to inhibitheparanase protein expression using antisense phosphorothioateoligonucleotides.

SUMMARY OF INVENTION

This invention provides an oligonucleotide having a sequencecomplementary to a sequence of a ribonucleic acid encoding a heparanase,wherein:

-   -   (a) the oligonucleotide hybridizes with the ribonucleic acid        under conditions of high stringency and is between 10 and 40        nucleotides in length;    -   (b) the internucleoside linkages of the oligonucleotide comprise        at least one phosphorothioate linkage; and    -   (c) hybridization of the oligonucleotide to the ribonucleic acid        inhibits expression of the heparanase, wherein inhibition of        heparanase expression means at least a 50% reduction in the        quantity of heparanase as follows: (a) a T24 bladder carcinoma        cell is exposed to a complex of the oligonucleotide and        lipofectin at an oligonucleotide concentration of 1 μM and a        lipofectin concentration of 10 μg/ml for 5 hours at 37° C., (b)        the complex is completely removed after such exposure, (c) 19        hours later the cell is scraped, washed and extracted in lysis        buffer, (d) the nucleus of the cell is removed by        centrifugation, (e) the cytoplasmic proteins in the resulting        supernatant are separated according to mass by sodium dodecyl        sulphate polyacrylamide gel electrophoresis, (f) the protein is        transferred to a polyvinylidene difluoride membrane that is        incubated at room temperature for 1-2 hours in incubation        solution (g) the membrane is exposed to 1 μg/ml of an antibody        directed against heparanase at 4° C. for 12 hours, (h) the        membrane is exposed to wash buffer and incubated for 1 hour at        room temperature in blocking buffer comprising a 1:3,000        dilution of a peroxidase-conjugated secondary antibody directed        against an epitope on the antibody directed against        heparanase, (i) the membrane is exposed to a chemiluminescent        cyclic diacylthydrazide and the oxidation of the cyclic        diacylthydrazide by the peroxidase is detected as a        chemiluminescent signal, and (j) the signal is quantitated by        laser-scanning densitometry as a measure of the amount of        heparanase expressed calculated as a percentage of heparanase        expression in an untreated cell.

This invention further provides the instant oligonucleotide, wherein theoligonucleotide comprises deoxyribonucleotides.

This invention further provides the instant oligonucleotide, wherein theoligonucleotide comprises ribonucleotides.

This invention further provides the instant oligonucleotide, whereinevery internucleoside linkage is a phosphorothioate linkage.

This invention further provides the instant oligonucleotide, wherein theoligonucleotide is between 15 and 25 nucleotides in length.

This invention further provides the instant oligonucleotide, wherein theoligonucleotide is about 20 nucleotides in length.

This invention further provides the instant oligonucleotide, wherein thesequence of the oligonucleotide is selected from the following: (a)CCCCAGGAGCAGCAGCAGCA; (SEQ ID NO:3) (b) GTCCAGGAGCAACTGAGCAT; (SEQ IDNO:4) and (c) AGGTGGACTTTCTTAGAAGT. (SEQ ID NO:5)This invention further provides the instant oligonucleotide, wherein theoligonucleotide further comprises a modified internucleoside linkage.

This invention further provides the instant oligonucleotide, wherein themodified internucleoside linkage is a peptide-nucleic acid linkage, amorpholino linkage, a phosphodiester linkage or a stereo-regularphosphorothioate.

This invention further provides the instant oligonucleotide, wherein theoligonucleotide further comprises a modified sugar moiety.

This invention further provides the instant oligonucleotide, wherein themodified sugar moiety is 2′-O-alkyl oligoribonucleotide.

This invention further provides the instant oligonucleotide, wherein theoligonucleotide further comprises a modified nucleobase.

This invention further provides the instant oligonucleotide, wherein themodified nucleobase is a 5-methylpyrimidine or a 5-propynyl pyrimidine.

This invention further provides the instant oligonucleotide, wherein theheparanase is a human heparanase.

This invention also provides a method of inhibiting expression of aheparanase in a cell comprising contacting the cell with the instantoligonucleotide under conditions such that the oligonucleotidehybridizes with mRNA encoding the heparanase so as to thereby inhibitthe expression of the heparanase.

This invention further provides the instant method, wherein the cell isa cancer cell.

This invention also provides a composition comprising the instantoligonucleotide in an amount effective to inhibit expression of aheparanase in a cell and a carrier.

This invention further provides the instant composition, wherein theoligonucleotide and the carrier are capable of passing through a cellmembrane.

This invention further provides the instant composition, wherein thecarrier comprises a membrane-permeable cationic reagent.

This invention further provides the instant composition, wherein thecationic reagent is lipofectin.

This invention also provides a method of treating a tumor in a subjectwhich comprises administering to the subject an amount of the instantoligonucleotide effective to inhibit expression of a heparanase in thesubject and thereby treat the tumor.

This invention further provides the instant method, wherein the subjectis a human being.

This invention further provides the instant method, wherein thetreatment of the tumor is effected by reducing tumor growth.

This invention further provides the instant method, wherein thetreatment of the tumor is effected by reducing tumor metastasis.

This invention further provides the instant method, wherein thetreatment of the tumor is effected by reducing angiogenesis.

This invention also provides a method of treating a subject whichcomprises administering to the subject an amount of the instantoligonucleotide effective to inhibit expression of a heparanase in thesubject and thereby treat the subject.

This invention further provides the instant method, wherein the subjectis a human being.

This invention also provides the use of the instant oligonucleotide forthe preparation of a pharmaceutical composition for treating a tumor ina subject which comprises admixing the oligonucleotide in an amounteffective to inhibit expression of a heparanase in the subject, with apharmaceutical carrier.

This invention also provides an oligonucleotide having a sequencecomplementary to a sequence of a ribonucleic acid encoding a heparanase,wherein:

-   -   (a) the oligonucleotide hybridizes with the ribonucleic acid        under conditions of high stringency and is between 10 and 40        nucleotides in length;    -   (b) the internucleoside linkages of the oligonucleotide comprise        at least one phosphorothioate linkage; and    -   (c) hybridization of the oligonucleotide to the ribonucleic acid        inhibits expression of the heparanase.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Western Blot showing reduction of heparanase protein expressionby LB85 (90% compared to untreated control) and LB90 (95% compared tocontrol). Also shown are other phosphorothioate oligonucleotides thatwere tested but had no significant effect on heparanase proteinexpression.

FIG. 2 Northern blot showing downregulation of heparanase proteinexpression by LB85, LB90 and LB65. Also shown are other phosphorothioateoligonucleotides that were ineffective at downregulating mRNA expressionas well as the G3DPH control.

FIG. 3 This figure shows the DNA sequence encoding Human heparanaseprotein.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are presented as an aid in understanding thisinvention: CDNA Complementary DNA; DNA Deoxyribonucleic Acid; DTT(−)-1,4-Dithio-L-Threitol; ER Endoplasmic Reticulum; G3PDHGlycerol-3-Phosphate Dehydrogenase. HepesN-(2-Hydroxyethyl)piperazine-N′-(2- ethanesulfonic acid) HIV HumanImmunodeficiency Virus; ICAM- 1 Intercellular Adhesion Molecule-1; IgImmunoglobulin; MEM Modified Eagle's Medium mRNA Messenger RNA; PBSPhosphate Buffered Saline; RNA Ribonucleic Acid; SDS Sodium DodecylSulphate; SSC Saline-Sodium Citrate Buffer; UV Ultra Violet.

“Administer” shall mean any of the various methods and delivery systemsknown to those skilled in the art. The administering can be performed,for example, orally, via implant, transmucosally, transdermally andsubcutaneously.

“Carrier” shall mean any of the various carriers known to those skilledin the art. “Pharmaceutical carrier” shall mean the same, excepting thatthe carrier shall be pharmaceutically acceptable. In one embodiment, thecarrier is a membrane-permeable cationic reagent, preferably lipofectin.In another embodiment, the carrier is a non-covalently linked peptidecomplex. In another embodiment the carrier is a covalent linked peptidecomplex, comprising, for example, a pH sensitive fusogenic peptide. Inother embodiments the carriers are polyamidodendrimers; transferrinpolylysine; polyglycolic acid co-polymers and any delivery in polymersthat can be used to nanoencapsulate, such as polylactic acid. In anotherembodiment the oligonucleotide is modifed by addition of a 5′cholesteryl; and in other embodiments by 5′ lipid or alkyl.

The following carriers are set forth, in relation to their most commonlyassociated delivery systems, by way of example, and do not precludecombinations of carriers.

Dermal delivery systems include, for example, aqueous and nonaqueousgels, creams, multiple emulsions, microemulsions, liposomes, ointments,aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon basesand powders, and can contain excipients such as solubilizers, permeationenhancers (e.g., fatty acids, fatty acid esters, fatty alcohols andamino acids), and hydrophilic polymers (e.g., polycarbophil andpolyvinylpyrolidone). In one embodiment, the pharmaceutically acceptablecarrier is a liposome or a transdermal enhancer. Examples of liposomeswhich can be used in this invention include the following: (1)CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipidN,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmityl-spermine anddioleoyl phosphatidylethanol-amine (DOPE) (GIBCO BRL); (2) CytofectinGSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (GlenResearch); (3) DOTAP(N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)(Boehringer Mannheim); and (4) Lipofectamine, 3:1 (M/M) liposomeformulation of the polycationic lipid DOSPA and the neutral lipid DOPE(GIBCO BRL).

Transmucosal delivery systems include patches, tablets, suppositories,pessaries, gels and creams, and can contain excipients such assolubilizers and enhancers (e.g., propylene glycol, bile salts and aminoacids), and other vehicles (e.g., polyethylene glycol, fatty acid estersand derivatives, and hydrophilic polymers such ashydroxypropylmethylcellulose and hyaluronic acid).

Injectable drug delivery systems include solutions, suspensions, gels,microspheres and polymeric injectables, and can comprise excipients suchas solubility-altering agents (e.g., ethanol, propylene glycol andsucrose) and polymers (e.g., polycaprolactones and PLGA's). Implantablesystems include rods and discs, and can contain excipients such as PLGAand polycaprolactone.

Oral delivery systems include tablets and capsules. These can containexcipients such as binders (e.g., hydroxypropylmethylcellulose,polyvinyl pyrrolidone, other cellulosic materials and starch), diluents(e.g., lactose and other sugars, starch, dicalcium phosphate andcellulosic materials), disintegrating agents (e.g., starch polymers andcellulosic materials) and lubricating agents (e.g., stearates and talc).

“Blocking buffer” shall mean 5% non-fat milk in phosphate bufferedsaline containing 0.5% Tween-20, wherein Tween-20 ispolyoxyethylene(20)sorbitan monolaurate.

“Centrifugation” shall mean centrifugation at 8,000 cpm for 10 min. at4° C.

“Chemiluminescent cyclic diacylthydrazide” shall mean Luminol(Amersham).

“Complex” shall mean, when applied to a “complex” of oligonucleotide andlipofectin, a solution comprising Lipofectin diluted in 100 μl ofOpti-MEM medium (Gibco BRL) to give a final concentration of 10 μg/ml,and phosphorothioate oligonucleotides diluted in 100 μl of Opti-MEM togive a final concentration of 1 μM mixed gently and preincubated at roomtemperature for 30 min to allow complexes to form and then diluted with800 μl of Opti-MEM media.

“Extracted in lysis buffer” shall mean exposing cells to 40-50 mL of 10mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 15 μg/ml aprotinin,leupeptin, 50 μg/ml Pefablock SC, 0.5 mM DTT, and 0.3% of Nonidet P40 at4° C. for 10 min.

“Heparanase” shall mean the protein encoded by the nucleotide sequenceshown in FIG. 3 (SEQ ID NO:17) and having the amino acid sequence SEQ IDNO:18, and any variants thereof, whether artificial or naturallyoccurring.

“Incubation solution” shall mean Blotto A (Amersham) [5% bovine serumalbumin in phosphate buffered saline containing 0.5% Tween-20, whereinTween-20 is polyoxyethylene(20)sorbitan monolaurate.]

“Inhibit” shall mean to slow, stop or otherwise impede.

“Modified nucleobase” shall mean, when applied to an oligonucleotide,nucleotide bases that are substituted or modified. Apart from the basesof adenine, guanine, cytosine, and thymine, other natural bases such asinosine, deoxyinosine, and hypoxanthine are acceptable in theoligonucleotide moiety useful in the subject invention. In addition,isosteric purine 2′deoxy-furanoside analogues, 2′-deoxynebularine or2′deoxyxanthosine, or other purine and pyrimidine analogues such as5-methylpyrimidine or a 5-propynyl pyrimidine may also be used.

“Modified sugar” shall mean, when applied to an oligonucleotide moiety,a sugar modified or replaced so as to be ribose, glucose, sucrose, orgalactose, or any other sugar. Alternatively, the oligonucleotide mayhave one or more of its sugars substituted or modified in its 2′position, i.e. 2′alkyl or 2′-O-alkyl. An example of a 2′-O-allyl sugaris a 2′-O-methylribonucleotide. Further, the oligonucleotide may haveone or more of its sugars substituted or modified to form an α-anomericsugar.

“Oligonucleotide” shall mean an oligonucleotide oroligodeoxyribonucleotide or an oligoribonucleotide.

“Phosphorothioate”, when applied to an oligonucleotide, shall mean anoligonucleotide in which a sulfur atom replaces one or more of thenon-bridging oxygen atoms in one or more phosphodiester linkages, i.e.an oligonucleotide having one or more phosphorothiodiester linkages.Each phosphorothiodiester linkage can occur as either an Rp or Spdiastereomer. A bridging oxygen atom is an oxygen atom in aphosphodiester linkage of a nucleic acid which joins phosphorous to asugar.

One or more of the phosphorothiodiester linkages of the oligonucleotidemoiety may be modified by replacing one or both of the two bridgingoxygen atoms of the linkage with analogues such as —NH, —CH2, or —S.Other oxygen analogues known in the art may also be used.

A phosphorothioate oligonucleotide may be stereo regular, stereonon-regular or stereo random. A stereo regular phosphorothioateoligonucleotide is a oligonucleotide in which all the phosphodiesterlinkages or phosphorothiodiester linkages polarize light in the samedirection. Each phosphorous in each linkage may be either an Sp or Rpdiastereomer. Phosphorothioate oligonucleotides which are created in anautomated synthesizer are stereo random which means that eachphosphorous atom in the phosphorothioate oligonucleotide has a 50%chance of being either an Sp or an Rp diastereomer.

“Specifically hybridize”, when referring to the action of the instantoligonucleotide on a target mRNA molecule, shall mean the annealing ofthe instant oligonucleotide to the target mRNA molecule, based onsequence complementarity, without annealing to another mRNA moleculelacking a sequence complementary to the instant oligonucleotide. Thepropensity for hybridization between nucleic acids depends on thetemperature and ionic strength of their milieu, the length of theoligonucleotide and the degree of complementarity. The effect of theseparameters on hybridization is well known in the art (see Sambrook,1989).

“Stringent conditions” or “Stringency”, shall refer to the conditionsfor hybridization as defined by the nucleic acid, salt, and temperature.These conditions are well known in the art and may be altered. Numerousequivalent conditions comprising either low or high stringency depend onfactors such as the length and nature of the sequence (DNA, RNA, basecomposition), nature of the target (DNA, RNA, base composition), milieu(in solution or immobilized on a solid substrate), concentration ofsalts and other components (e.g., formamide, dextran sulfate and/orpolyethylene glycol), and temperature of the reactions (within a rangefrom about 5° C. below the melting temperature of the probe to about 20°C. to 25° C. below the melting temperature). One or more factors be maybe varied to generate conditions of either low or high stringencydifferent from, but equivalent to, the above listed conditions. As willbe understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences.

“Subject” shall mean any animal, such as a human, a primate, a mouse, arat, a guinea pig or a rabbit.

“Variants” shall mean proteins having at least 80%, preferably at least90%, more preferably at least 95% similarity with SEQ ID NO:18. As usedherein “similar” is used to denote which sequences when aligned havesimilar (identical or conservatively substituted) amino acids in likepositions or regions, where identical or conservatively replaced aminoacids are those which do not alter the activity or function of theprotein as compared to the starting protein.

“Wash buffer” shall mean phosphate buffered saline with 0.5% Tween-20,wherein Tween-20 is polyoxyethylene(20)sorbitan monolaurate.

“Washed” shall mean washed in phosphate buffered saline.

Having due regard to the preceding definitions, the present inventionprovides an oligonucleotide having a sequence complementary to asequence of a ribonucleic acid encoding a heparanase, wherein:

-   -   (a) the oligonucleotide hybridizes with the ribonucleic acid        under conditions of high stringency and is between 10 and 40        nucleotides in length;    -   (b) the internucleoside linkages of the oligonucleotide comprise        at least one phosphorothioate linkage; and    -   (c) hybridization of the oligonucleotide to the ribonucleic acid        inhibits expression of the heparanase, wherein inhibition of        heparanase expression means at least a 50% reduction in the        quantity of heparanase as follows: (a) a T24 bladder carcinoma        cell is exposed to a complex of the oligonucleotide and        lipofectin at an oligonucleotide concentration of 1 μM and a        lipofectin concentration of 10 μg/ml for 5 hours at 37° C., (b)        the complex is completely removed after such exposure, (c) 19        hours later the cell is scraped, washed and extracted in lysis        buffer, (d) the nucleus of the cell is removed by        centrifugation, (e) the cytoplasmic proteins in the resulting        supernatant are separated according to mass by sodium dodecyl        sulphate polyacrylamide gel electrophoresis, (f) the protein is        transferred to a polyvinylidene difluoride membrane that is        incubated at room temperature for 1-2 hours in incubation        solution (g) the membrane is exposed to 1 μg/ml of an antibody        directed against heparanase at 4° C. for 12 hours, (h) the        membrane is exposed to wash buffer and incubated for 1 hour at        room temperature in blocking buffer comprising a 1:3,000        dilution of a peroxidase-conjugated secondary antibody directed        against an epitope on the antibody directed against        heparanase, (i) the membrane is exposed to a chemiluminescent        cyclic diacylthydrazide and the oxidation of the cyclic        diacylthydrazide by the peroxidase is detected as a        chemiluminescent signal, and (j) the signal is quantitated by        laser-scanning densitometry as a measure of the amount of        heparanase expressed calculated as a percentage of heparanase        expression in an untreated cell.

In one embodiment every internucleoside linkage is a phosphorothioatelinkage. In one embodiment the ribonucleic acid molecule is a messengerribonucleic acid molecule. In a further embodiment the ribonucleic acidmolecule encodes for heparanase protein. In one embodiment thehybridization of the oligonucleotide to the ribonucleic acid moleculeinhibits heparanase protein expression. In one embodiment the heparanaseprotein is human. In one embodiment the oligonucleotide comprisesdeoxyribonucleotides. In another embodiment the oligonucleotidecomprises ribonucleotides. In one embodiment the oligonucleotidesequence is a minimum of 10 and a maximum of 40 nucleobases in length.In another embodiment the oligonucleotide sequence is a minimum of 15and a maximum of 25 nucleobases in length. In the preferred embodimentthe phosphorothioate oligonucleotide is about 20 nucleobases in length.

This invention further provides the instant oligonucleotide, wherein thesequence is selected from the following group: (a) CCCCAGGAGCAGCAGCAGCA;(SEQ ID NO:3) (b) GTCCAGGAGCAACTGAGCAT; (SEQ ID NO:4) and (c)AGGTGGACTTTCTTAGAAGT. (SEQ ID NO:5)

In one embodiment hybridization occurs at target residues 137-156 of theinstant heparanase mRNA molecule (see SEQ ID NO:17) for SEQ ID NO:3,residues 707-726 for SEQ ID NO:4 and residues 852-871 for SEQ ID NO:5.

This invention further provides the instant oligonucleotide, wherein theoligonucleotide further comprises a modified internucleoside linkage. Indifferent embodiments the modified internucleoside linkage is apeptide-nucleic acid linkage, a morpholino linkage, a phosphodiesterlinkage or a stereo-regular phosphorothioate.

This invention further provides the instant oligonucleotide, wherein theoligonucleotide further comprises a modified sugar moiety. In oneembodiment the modified sugar moiety is 2′-O-alkyl oligoribonucleotide.

This invention further provides the instant oligonucleotide sequence,wherein the sequence further comprises a modified nucleobase. In oneembodiment the modified nucleobase is a 5-methylpyrimidine. In oneembodiment the modified nucleobase is a 5-propynyl pyrimidine.

This invention also provides a method of inhibiting expression of aheparanase in a cell comprising contacting the cell with the instantoligonucleotide under conditions such that the oligonucleotidehybridizes with mRNA encoding the heparanase so as to thereby inhibitthe expression of the heparanase. In one embodiment the cell is amammalian cell. In a further embodiment the cell is a human cell. In thepreferred embodiment the cell is a cancer cell.

This invention also provides a composition comprising the instantoligonucleotide in an amount effective to inhibit expression of aheparanase in a cell and a carrier. In one embodiment the instantcomposition is capable of passing through a cell membrane. In oneembodiment the instant composition comprises a membrane-permeablecationic reagent. In one embodiment the instant reagent is lipofectin.In one embodiment the cell is a mammalian cell. In a further embodimentthe cell is a cancer cell.

In a further embodiment the cell is a human cell.

This invention also provides method of treating a tumor in a subjectwhich comprises administering to the subject an amount of the instantoligonucleotide effective to inhibit expression of a heparanase in thesubject and thereby treat the tumor. In the preferred embodiment thesubject is human. In one embodiment the tumor is treated by effecting areduction in tumor growth. In one embodiment the tumor is treated byeffecting a reduction in tumor metastasis. In one embodiment the tumoris treated by effecting a reduction in angiogenesis.

This invention also provides a method of treating a subject whichcomprises administering to the subject an amount of the instantoligonucleotide effective to inhibit expression of a heparanase in thesubject and thereby treat the subject.

In one embodiment the subject has an abnormality that is treated byinhibiting heparanase expression. In one embodiment the subject has anabnormality that is treated by inhibiting angiogenesis. In the preferredembodiment the subject is human.

This invention also provides the use of the instant oligonucleotide forthe preparation of a pharmaceutical composition for treating a tumor ina subject which comprises admixing the oligonucleotide in an amounteffective to inhibit expression of a heparanase in the subject, with apharmaceutical carrier. In the preferred embodiment the subject ishuman.

This invention also provides an oligonucleotide having a sequencecomplementary to a sequence of a ribonucleic acid encoding a heparanase,wherein:

-   -   (a) the oligonucleotide hybridizes with the ribonucleic acid        under conditions of high stringency and is between 10 and 40        nucleotides in length;    -   (b) the internucleoside linkages of the oligonucleotide comprise        at least one phosphorothioate linkage; and    -   (c) hybridization of the oligonucleotide to the ribonucleic acid        inhibits expression of the heparanase. In one embodiment        inhibition of heparanase expression means at least a 50%        reduction in the quantity of heparanase in particular as        follows: (a) a T24 bladder carcinoma cell is exposed to a        complex of the oligonucleotide and lipofectin at an        oligonucleotide concentration of about 1 μM and a lipofectin        concentration of about 10 μg/ml for about 5 hours at 37° C., (b)        the complex is completely removed after such exposure, (c) about        19 hours later the cell is scraped, washed and extracted in        lysis buffer, (d) the nucleus of the cell is removed by        centrifugation, (e) the cytoplasmic proteins in the resulting        supernatant are separated according to mass by sodium dodecyl        sulphate polyacrylamide gel electrophoresis, (f) the protein is        transferred to a polyvinylidene difluoride membrane that is        incubated at room temperature for about 1-2 hours in incubation        solution (g) the membrane is exposed to about 1 μg/ml of an        antibody directed against heparanase at 4° C. for about 12        hours, (h) the membrane is exposed to wash buffer and incubated        for about 1 hour at room temperature in blocking buffer        comprising a 1:3,000 dilution of a peroxidase-conjugated        secondary antibody directed against an epitope on the antibody        directed against heparanase, (i) the membrane is exposed to a        chemiluminescent cyclic diacylthydrazide and the oxidation of        the cyclic diacylthydrazide by the peroxidase is detected as a        chemiluminescent signal, and (j) the signal is quantitated by        laser-scanning densitometry as a measure of the amount of        heparanase expressed calculated as a percentage of heparanase        expression in an untreated cell.

The following Experimental Details are set forth to aid in anunderstanding of the invention, and are not intended, and should not beconstrued, to limit in any way the invention set forth in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS

Antisense phosphorothioate oligonucleotides inhibit heparanase proteinexpression: Inhibition of heparanase protein expression byanti-heparanase phosphorothioate oligonucleotides was demonstrated byWestern blotting, as is shown in FIG. 1. T24 bladder carcinoma cellswere treated with the phosphorothioate oligonucleotides complexed withLipofectin. The optimum concentration of the oligomer was 1 μM. Theoptimum concentrations of Lipofectin were 10 and 12.5 μg/ml. The optimumincubation time of the cells with complexes was 5 h, and expression ofheparanase protein was assayed after a further 19 h of incubation incomplete media in the absence of complex. The most active sequences wereLB 85 (SEQ ID NO:4), 90 (SEQ ID NO:5) and 65 (SEQ ID NO:3). Theexpression of heparanase protein for LB85, 90 and 65 was reduced byapproximately 90, 95 and 75%, respectively (FIG. 1). No other oligomersproduced any significant reduction in target protein levels.

Downregulation of Heparanase protein mRNA by antisense heparanasephosphorothioate oligonucleotides: Heparanase protein mRNA expression inT24 cells treated with anti-heparanase phosphorothioate oligonucleotideswas evaluated by Northern blotting, using the heparanase cDNA fragmentas a probe. The 4.4 and 2.0 kB Heparanase protein mRNA species wereeasily visualized (FIG. 2). Significantly, only the phosphorothioateoligonucleotides that demonstrated activity in the Western blot wereactive in the Northern blot. None of the four control phosphorothioateoligonucleotides (LB78—SEQ ID NO:7, LB88—SEQ ID NO:8, LB94—SEQ ID NO:6,LB101—SEQ ID NO:9), which were entirely inactive in the Western blot,demonstrated any activity vs. the untreated controls in the Northernblot. The extent of downregulation for LB90 (SEQ ID NO:5) wasapproximately 90%, and for LB65 (SEQ ID NO:3) and LB85 (SEQ ID NO:4)approximately 75% (FIG. 2). These results confirm the Western analysis,and demonstrate that these phosphorothioate oligonucleotides cause asequence specific anti-heparanase effect.

The Heparanase protein mRNA blots were then stripped and reprobed with acontrol glycerol-3-phosphate dehydrogenase cDNA probe (G3PDH). The datademonstrated essentially equal RNA loading in each lane, and no decreasein levels of this mRNA species.

Heparan Sulfate Fragment Release Assay: We examined ability of lysatesof DU145 prostate cancer cells to release tritiated heparan sulfatefragments from tritiated heparan sulfate proteoglycan. DU145 cells weretreated for 5 hrs in opti-MEM media with a 1 micromolar concentration ofLB90 (or control oligomer LB101—SEQ ID NO:9—that does not downregulateheparanase expression by Western blotting)+10 micrograms/mL Lipofectin(each oligomer). At the end of this time, the oligonucleotides wasremoved, and complete media added. This procedure was repeated for threeconsecutive days. After 72 hours total incubation at 37° C., the cellswere lysed in buffer, and the lysates incubated with tritiated heparansulfate proteoglycan. The tritiated heparan sulfate fragments wereadsorbed onto filters and counted. Experiments were performed induplicate. LB90 (SEQ ID NO:5), as vs. LB101, downregulated the activityof intracellular heparanase by 70% as measured by the release oftritiated heparan sulfate. Treatment of DU145 cells as above, but foronly two days, led to a 60% decrease in tritiated heparan sulfaterelease, while treatment for only one day led to a 50% decrease inrelease.

Materials and Methods

Cells: T24 cells were grown at 37° C. in a humidified 5% CO₂ incubatorin McCoy's 5A medium (Gibco BRL, Grand Island, N.Y.), containing 10%(v/v) heat inactivated (56° C.) fetal bovine serum (FBS) (Gibco BRL,Grand Island, N.Y.), supplemented with 25 mM Hepes, 100 units/mLpenicillin G sodium and 100 g/mL streptomycin sulfate.

Reagents: Anti-human heparanase mouse monoclonal antibody HP-130 (IgG1subclass) were obtained from Insight (Rehovot, Israel). Anti-mousehorseradish peroxidase conjugated secondary antibody was from Amersham,Arlington Heights, Ill. Lipofectin was purchased from Gibco BRL.

Synthesis of phosphorothioate oligonucleotides: The all-phosphorothioateoligonucleotides used in these studies were synthesized on an AppliedBiosystems (Foster City, Calif.) model 380B DNA synthesizer by standardmethods. Sulfurization was performed using tetraethylthiuramdisulfide/acetonitrile. Following cleavage from controlled pore glasssupport, oligodeoxynucleotides were base deblocked in ammonium hydroxideat 60° C. for 8 h and purified by reversed-phase HPLC [0.1Mtriethylammonium bicarbonate/acetonitrile; PRP-1 support]. Oligomerswere detritylated in 3% acetic acid and precipitated with 2%lithiumperchlorate/acetone, dissolved in sterile water andreprecipitated as the sodium salt from 1 M NaCl/ethanol. Concentrationsof the full length species were determined by UV spectroscopy.

The sequences of the phosphorothioate oligonucleotides used were (5′ to3′): TGGGCTCACCTGGCTGCTCC (LB63); SEQ ID NO:1 CGCCAGCTGCCGCGCAGCGG(LB62); SEQ ID NO:2 CCCCAGGAGCAGCAGCAGCA (LB65); SEQ ID NO:3GTCCAGGAGCAACTGAGCAT (LB85); SEQ ID NO:4 AGGTGGACTTTCTTAGAAGT (LB90);SEQ ID NO:5 TCAAATAGTAGTGATGCCAT (LB94); SEQ ID NO:6CTTCTCCTCCACATCAGGAG (LB78); SEQ ID NO:7 ATTGATGAAAATATCAGCCT (LB88);SEQ ID NO:8 TTATCCAGCCACATAAAGCC (LB101); SEQ ID NO:9AGCGCAGGCTTCGAGCGCAG (LB64); SEQ ID NO:10 GATAGCCAATAATCAGGTAA (LB105);SEQ ID NO:11 GGTGCCACCAAACCTCAGGT (LB74); SEQ ID NO:12GAGCCCCAGCGCCCTTTTCT (LB60); SEQ ID NO:13 GGAGAACCCAGGAGGATGAG (LB72);SEQ ID NO:14 CTACAGAGCTTCTTGAGTAG (LB81); SEQ ID NO:15 andTATACCTTGGATTGTCAGTG (LB109). SEQ ID NO:16

Treatment of cells with phosphorothioate oligonucleotide/Lipofectincomplexes: Cells were seeded the day before the experiment in 6-wellplates at a density of 25-30×10⁴ cells per well to be 65-70% confluenton the day of the experiment. The transfections were performed inOpti-MEM medium (Gibco BRL) as per the manufacturer's instructions.Lipofectin was diluted in 100 μl of Opti-MEM medium to give a finalconcentration of 10 or 12.5 μg/ml, and phosphorothioate oligonucleotideswere diluted in 100 μl of Opti-MEM to give a final concentration of 1μM. The solutions were mixed gently and preincubated at room temperaturefor 30 min to allow complexes to form. Then, 800 μl of Opti-MEM mediawas added, the solution mixed, and overlaid on the cells that werepre-washed with Opti-MEM. The cells were then incubated at 37° C. for 5h, re-fed with fresh complete media containing 10% FBS (nophosphorothioate oligonucleotide/Lipofectin complexes present), andallowed to incubate for an additional 19 h before cell lysis and extractpreparation.

Western Blot Analysis: Cells treated with phosphorothioateoligonucleotide-lipid complexes were scraped, washed with cold PBS andthen extracted in 40-50 mL of 10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mMMgCl₂, 15 μg/ml aprotinin, leupeptin, 50 μg/ml Pefablock SC, 0.5 mM DTT,and 0.3% of Nonidet-P40 at 4° C. for 10 min. The nuclei were removed bycentrifugation for 10 min at 4° C., and cytoplasmic proteinconcentrations in the supernatants were determined using the Bio-Radprotein assay system (Bio-Rad Laboratories, Richmond, Calif.). Aliquotsof cytoplasmic extracts, containing 35-40 μg of protein, were resolvedby 10% PAGE. Proteins were then transferred to PVDF membranes (Amersham,Arlington Heights, Ill.), and the filters incubated at room temperaturefor 1-2 h in Blotto A [5% BSA in PBS containing 0.5% Tween 20]. Thefilters were then probed with 1 μg/ml of the anti-heparanase antibody inBlotto A at 4° C. overnight. After washing in PBS-0.5% Tween 20 buffer(3×7 min, room temperature), the filters were incubated for 1 h at roomtemperature in 5% non-fat milk in PBS containing 0.5% Tween-20 with a1:3,000 dilution of a peroxidase-conjugated secondary antibody(Amersham). After washing (3×10 min), ECL was performed according to themanufacturer's instructions. Protein expression was quantitativelyanalyzed via laser-scanning densitometry using NIH Image Version 1.61software. All results were calculated as a percentage of proteinexpression in treated vs. untreated cells.

Northern Blot Analysis: Total cellular RNA was isolated using TRIZOLReagent (Gibco BRL). 20-30 μg was resolved on a 1.2% agarose gelcontaining 1.1% formaldehyde and transferred to Hybond-N nylon membranes(Amersham). A human heparanase cDNA probe (kindly provided by Insight,Rehovot, Israel) was ³²P-radiolabeled with [-³²P]dCTP by random primerlabeling using a commercially available kit (Promega) according to themanufacturer's instructions. The blots were then hybridized with thecDNA probe in 50% formamide, 5×SSC, 5× Denhard's solution, 0.5% SDS, 1%dextran sulfate, and 0.1 mg/ml of salmon sperm DNA overnight at 42° C.The filters were washed at room temperature, twice for 15 min in 2×SSCand 0.1% SDS, once for 20 min in 1×SSC and 0.1% SDS, and finally twicefor 15 min in 0.1×SSC and 0.1% SDS at 65° C. The filters were exposed toKodak X-ray film for 12-48 h with intensifying screens at −70° C., andthen developed. A similar procedure was repeated for the G3PDH control.

REFERENCES

-   1. Kussie, P. H. et al. (1999) Biochem. Biophys. Res. Comm.    261:183-187.-   2. Lebedeva et al, (2000) Eur J Pharm Biopharm. 2000    July;50(1):101-19.-   3. Sambrook et al., “Molecular Cloning: A Laboratory Manual”, Second    Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y.-   4. Vlodasky, I. et al, (1999) Nature Medicine 5:793-802.

1. An oligonucleotide having a sequence complementary to a sequence of aribonucleic acid encoding a heparanase, wherein: (a) the oligonucleotidehybridizes with the ribonucleic acid under conditions of high stringencyand is between 10 and 40 nucleotides in length; (b) the internucleosidelinkages of the oligonucleotide comprise at least one phosphorothioatelinkage; and (c) hybridization of the oligonucleotide to the ribonucleicacid inhibits expression of the heparanase, wherein inhibition ofheparanase expression means at least a 50% reduction in the quantity ofheparanase as follows: (a) a T24 bladder carcinoma cell is exposed to acomplex of the oligonucleotide and lipofectin at an oligonucleotideconcentration of 1 μM and a lipofectin concentration of 10 μg/ml for 5hours at 37° C., (b) the complex is completely removed after suchexposure, (c) 19 hours later the cell is scraped, washed and extractedin lysis buffer, (d) the nucleus of the cell is removed bycentrifugation, (e) the cytoplasmic proteins in the resultingsupernatant are separated according to mass by sodium dodecyl sulphatepolyacrylamide gel electrophoresis, (f) the protein is transferred to apolyvinylidene difluoride membrane that is incubated at room temperaturefor 1-2 hours in incubation solution (g) the membrane is exposed to 1μg/ml of an antibody directed against heparanase at 4° C. for 12 hours,(h) the membrane is exposed to wash buffer and incubated for 1 hour atroom temperature in blocking buffer comprising a 1:3,000 dilution of aperoxidase-conjugated secondary antibody directed against an epitope onthe antibody directed against heparanase, (i) the membrane is exposed toa chemiluminescent cyclic diacylthydrazide and the oxidation of thecyclic diacylthydrazide by the peroxidase is detected as achemiluminescent signal, and (j) the signal is quantitated bylaser-scanning densitometry as a measure of the amount of heparanaseexpressed calculated as a percentage of heparanase expression in anuntreated cell.
 2. The oligonucleotide of claim 1, wherein theoligonucleotide comprises deoxyribonucleotides.
 3. The oligonucleotideof claim 1, wherein the oligonucleotide comprises ribonucleotides. 4.The oligonucleotide of claim 1, wherein every internucleoside linkage isa phosphorothioate linkage.
 5. The oligonucleotide of claim 1, whereinthe oligonucleotide is between 15 and 25 nucleotides in length.
 6. Theoligonucleotide of claim 1, wherein the oligonucleotide is about 20nucleotides in length.
 7. The oligonucleotide of claim 1, wherein thesequence of the oligonucleotide is selected from the following: (a)CCCCAGGAGCAGCAGCAGCA; (SEQ ID NO:3) (b) GTCCAGGAGCAACTGAGCAT; (SEQ IDNO:4) and (c) AGGTGGACTTTCTTAGAAGT. (SEQ ID NO:5)


8. The oligonucleotide of claim 1, wherein the oligonucleotide furthercomprises a modified internucleoside linkage.
 9. The oligonucleotide ofclaim 8, wherein the modified internucleoside linkage is apeptide-nucleic acid linkage, a morpholino linkage, a phosphodiesterlinkage or a stereo-regular phosphorothioate.
 10. The oligonucleotide ofclaim 1, wherein the oligonucleotide further comprises a modified sugarmoiety.
 11. The oligonucleotide of claim 10, wherein the modified sugarmoiety is 2′-O-alkyl oligoribonucleotide.
 12. The oligonucleotide ofclaim 1, wherein the oligonucleotide further comprises a modifiednucleobase.
 13. The oligonucleotide of claim 12, wherein the modifiednucleobase is a 5-methylpyrimidine or a 5-propynyl pyrimidine.
 14. Theoligonucleotide of claim 1, wherein the heparanase is a humanheparanase.
 15. A method of inhibiting expression of a heparanase in acell comprising contacting the cell with the oligonucleotide of claim 1under conditions such that the oligonucleotide hybridizes with mRNAencoding the heparanase so as to thereby inhibit the expression of theheparanase.
 16. The method of claim 15, wherein the cell is a cancercell.
 17. A composition comprising the oligonucleotide of claim 1 in anamount effective to inhibit expression of a heparanase in a cell and acarrier.
 18. The composition of claim 17, wherein the oligonucleotideand the carrier are capable of passing through a cell membrane.
 19. Thecomposition of claim 18, wherein the carrier comprises amembrane-permeable cationic reagent.
 20. The composition of claim 19,wherein the cationic reagent is lipofectin.
 21. A method of treating atumor in a subject which comprises administering to the subject anamount of the oligonucleotide of claim 1 effective to inhibit expressionof a heparanase in the subject and thereby treat the tumor.
 22. A methodof treating a subject which comprises administering to the subject anamount of the oligonucleotide of claim 1 effective to inhibit expressionof a heparanase in the subject and thereby treat the subject.
 23. Themethod of claim 21 or 22, wherein the subject is a human being.
 24. Themethod of claim 21, wherein the treatment of the tumor is effected byreducing tumor growth.
 25. The method of claim 21, wherein the treatmentof the tumor is effected by reducing tumor metastasis.
 26. The method ofclaim 21, wherein the treatment of the tumor is effected by reducingangiogenesis.
 27. Use of the oligonucleotide of claim 1 for thepreparation of a pharmaceutical composition for treating a tumor in asubject which comprises admixing the oligonucleotide in an amounteffective to inhibit expression of a heparanase in the subject, with apharmaceutical carrier.
 28. An oligonucleotide having a sequencecomplementary to a sequence of a ribonucleic acid encoding a heparanase,wherein: (a) the oligonucleotide hybridizes with the ribonucleic acidunder conditions of high stringency and is between 10 and 40 nucleotidesin length; (b) the internucleoside linkages of the oligonucleotidecomprise at least one phosphorothioate linkage; and (c) hybridization ofthe oligonucleotide to the ribonucleic acid inhibits expression of theheparanase.